High-Fidelity PCR Kits

Overview

Sso7d, a 7 kD protein obtained from Sulfolobus solfataricus, binds to dsDNA without any sequence specificity.

Sso7d is covalently linked to a DNA polymerase and is able to stabilize the polymerase-template complex without compromising the structural integrity, thermal stability, or catalytic activity of the enzyme.

The unique structure of this Sso7d fusion polymerase provides several significant advantages over ordinary DNA polymerase:

Comparison of common thermostable DNA polymerases. iProof Polymerase exhibits the highest fidelity of any commercially available thermostable polymerase, >50-fold higher than ordinary Taq polymerase. A common high-fidelity polymerase was ~8-fold higher than Taq, and an ultrahigh-fidelity polymerase was ~32-fold higher.

iProof Polymerase demonstrates unrivaled speed, leading to dramatically shorter overall reaction times. The reaction protocol for iProof Polymerase was compared to the recommended protocols for two competing polymerases. Each protocol was designed to amplify 1, 8, or 15 kb products in 30 cycles. Reactions with iProof Polymerase used a two-step protocol with a combined annealing and extension step, while the other reactions used three-step protocols with the minimum recommended extension times. Overall reaction times include temperature ramping times.

Comparison of amplification using iProof Polymerase and another supplier's fast- cycling enzyme. Manufacturer's recommended cycling conditions were used for both enzymes. Left, a 1 kb fragment was amplified from 10 ng human genomic DNA using 20 U/ml of iProof Polymerase (lanes 6–9) or 50 U/ml of the other enzyme (lanes 20–24) in a standard-ramping thermal cycler. Total run time for protocols using a 30 sec annealing/extension step was 48 min (lanes 6, 7, 20, and 21). Total run time for protocols using a 15 sec annealing/extension step was 39 min (lanes 8 and 9) or 40 min (lanes 22 and 23). Lane 5, 500 bp EZ Load marker. Right, the same 1 kb fragment was amplified from 100 ng human genomic DNA on a fast-ramping thermal cycler, using 50 U/ml of iProof Polymerase (lane 3) or 100 U/ml of the other enzyme (lane 4), and using an annealing/extension step of 30 sec. Total run time on the fast ramping cycler was 30 min. Lane 2, 500 bp EZ Load Marker.

Comparison of amplification using iProof Polymerase and another supplier's fast- cycling enzyme. Manufacturer's recommended cycling conditions were used for both enzymes. Left, a 1 kb fragment was amplified from 10 ng human genomic DNA using 20 U/ml of iProof Polymerase (lanes 6–9) or 50 U/ml of the other enzyme (lanes 20–24) in a standard-ramping thermal cycler. Total run time for protocols using a 30 sec annealing/extension step was 48 min (lanes 6, 7, 20, and 21). Total run time for protocols using a 15 sec annealing/extension step was 39 min (lanes 8 and 9) or 40 min (lanes 22 and 23). Lane 5, 500 bp EZ Load marker. Right, the same 1 kb fragment was amplified from 100 ng human genomic DNA on a fast-ramping thermal cycler, using 50 U/ml of iProof Polymerase (lane 3) or 100 U/ml of the other enzyme (lane 4), and using an annealing/extension step of 30 sec. Total run time on the fast ramping cycler was 30 min. Lane 2, 500 bp EZ Load Marker.

iProof Polymerase demonstrates increased speed with minimal amounts of enzyme. The gel images below compare the performance of three thermostable polymerases in amplifying a 1 kb DNA fragment using a two-step protocol that combined annealing and extension times ranging from 7 to 240 sec. iProof Polymerase successfully amplified the target with the shortest extension time (15 sec). Competing enzymes required an annealing/extension step of 30–60 sec or more and 5- to 10-fold more enzyme to produce comparable yields.

Annealing/extension time, sec

iProof High-Fidelity Polymerase

Competitor High-Fidelity Polymerase

Taq Polymerase

iProof DNA Polymerase delivers high yields with significantly less enzyme. A 1 kb fragment was amplified from 1.25 pM of lambda DNA template in 20 cycles, using combined annealing/extension step of variable duration as indicated. All enzymes were used according to manufacturer recommendations.

iProof™ High-Fidelity DNA Polymerase is a unique Pyrococcus-like proofreading enzyme fused to the Sso7d dsDNA-binding protein to create a thermostable fusion polymerase that accurately amplifies long products from a variety of DNA templates.

Features and Benefits of iProof High-Fidelity DNA Polymerase:

High fidelity — iProof DNA polymerase is 52-fold more accurate than Taq polymerase

Supporting Documentation

iProof™ Enzyme, Kit, and Buffers: guaranteed for 6 months at –20°C in a constant-temperature freezeriProof Master Mixes: guaranteed for 3 months at –20°C in a constant-temperature freezer (multiple freeze/thaws not recommended)

Storage at +4°C

Guaranteed for 3 months at 4°C

What is the education discount policy?

For more than 15 years, Bio-Rad has made science education a major priority. To support this effort, the company has implemented a discount policy that allows high school and college teaching laboratories to purchase kits, instruments, reagents, and other equipment at preferred prices.

How do I apply for the education discount?

If you are an educator at the high school or college level, visit our Education Discount Policy page to establish an education account number.

If you are placing an order, you may proceed with your order; the account price will be applied if it is lower than the list price. If the problem persists, please call customer service at
or send an email to